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Recombinant arthrobotrys oligospora of Aozl gene dual promoters and preparation method of recombinant arthrobotrys oligospora

A technology of Arthrospora oligospora and double promoters, which is applied in the biological field, can solve problems such as the decrease of the activity of predatory nematodes, and achieve the effects of improved activity of predatory nematodes, enhanced transcription level, and good genetic stability.

Inactive Publication Date: 2018-09-07
SHIHEZI UNIVERSITY
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Problems solved by technology

However, although Arthrobuspora oligospora isolated from nature showed strong nematode-predating activity at the initial stage of isolation, with the increase in the number of subcultures in the medium, the expression level of extracellular proteolytic enzymes also decreased, and the nematode-predating activity will gradually decrease, which has become a bottleneck restricting the development of efficient nematode-predating biological agents

Method used

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  • Recombinant arthrobotrys oligospora of Aozl gene dual promoters and preparation method of recombinant arthrobotrys oligospora
  • Recombinant arthrobotrys oligospora of Aozl gene dual promoters and preparation method of recombinant arthrobotrys oligospora
  • Recombinant arthrobotrys oligospora of Aozl gene dual promoters and preparation method of recombinant arthrobotrys oligospora

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Embodiment Construction

[0078] The present invention will be further described in detail below through specific examples.

[0079] 1. Cultivation of Arthrobuspora oligosporum.

[0080] The conidia of Arthrobuspora oligospora Xinjiang isolate XA3 (A.oligospora XA3) were inoculated on 0.4 g / mL corn flour agar medium (CMA), and cultured in a 25°C incubator ( figure 1 ). Cut 5-10 small pieces of the above-mentioned cultured mycelium into a sterilized corn grain conical flask, cultivate at 25°C for 21 days, and wash out the conidia with sterilized distilled water ( figure 2 ), and stored in a refrigerator at 4°C after counting.

[0081] 2. Cloning and sequence feature analysis of the 5' upstream sequence of Arthrobuspora oligospora Aoz1 gene.

[0082] ① Primer design for GenomeWalker of Arthrobuspora oligospora.

[0083] According to the Universal GenomeWalker produced by BD TM 2.0 Adapter primer sequences in the kit AP1: 5'-GTAATACGACTCACTATAGGGC-3' and AP2: 5'-ACTATAGGGCACGCGTGGT-3'; use the cDNA...

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Abstract

The invention discloses recombinant arthrobotrys oligospora of Aozl gene dual promoters, which is used for preventing and controlling ruminant animal digestive tract nematode diseases caused by nemathelminthes nematode, and a preparation method of the recombinant arthrobotrys oligospora. The Aozl gene promoters of arthrobotrys oligospora are cloned by adopting a chromosome DNA (Deoxyribonucleic Acid) walking technology, and the recombinant arthrobotrys oligospora efficiently expressing the Aozl gene dual promoters is constructed by utilizing an agrobacterium tumefaciens-mediated fungus transformation technology. Compared with a traditional genetic transformation method, the method provided by the invention has the advantages of wide recipient material range, high transformation efficiency,stable inheritance transformant and the like, and the bottleneck of a traditional transformation method is overcome. Compared with a non-recombinant maternal arthrobotrys oligospora strain, the strain obtained by the method has good genetic stability; the Aozl gene transcriptional level is enhanced and the nematode-trapping activity is improved; an efficient gene engineering strain is provided for researches and development of a biological prevention and control preparation for the ruminant animal digestive tract nematode diseases.

Description

technical field [0001] The invention relates to Arthrobuspora oligospora serine protease Aoz1 gene dual-promoter recombinant Arthrobuspora oligospora and a preparation method thereof, belonging to the field of biotechnology. technical background [0002] Ruminant digestive nematode disease is a kind of parasitic disease caused by various nematodes belonging to the nematode class Nematoda parasitizing in the digestive tract of ruminants, which seriously endangers the sustainable and healthy development of my country's aquaculture industry. According to statistics, among ruminant helminth diseases, digestive tract nematode infection accounts for more than half, among which Trichostrongylus, Haemonchus, Leptospirosis, Oster, Marshall, and Trichocerus Mainly. In recent years, with the continuous strengthening of the country's prevention and control measures for animal infectious diseases, the infectious diseases of ruminants have been effectively controlled, and the hazards of ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/113C12N15/10C12N1/15C12Q1/6897G01N33/53C12R1/645
CPCC12N9/58C12N15/10C12N15/80C12Q1/6897C12Y304/21014G01N33/5308C12Q2531/113C12Q2525/191C12Q2521/301
Inventor 孟庆玲乔军贡莎莎黄运福钟文强陈双庆刘昱成张星星蔡扩军王熙凤张凯陈英
Owner SHIHEZI UNIVERSITY
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